For a variety of clinical reasons it is desirable to quantatively assay one or more phenylethanolamines found, for example, in urine, in plasma, in brain tissue or the like. For example, the diagnosis of pheochromocytoma has been based upon measurement of the increased plasma or urinary levels of catecholamines and their metabolites such as metanephrine, normetanephrine and 3-methoxy-4-hydroxy-mandelic acid. Such diagnosis is important because these tumors produce a potentially curable form of hypertension, which otherwise is fatal. However, techniques for measurements of urinary catecholamines and catecholamine metabolites are non-specific, laborious, tedious and frequently associated with false positive results. Furthermore, plasma catecholamines may be within normal range, because some tumors may secrete large amounts of pharmacologically inactive metabolites (normetanephrine), but relatively small amounts of free norepinephrine and epinephrine. The measurement of the catecholamine metabolite normetanephrine in plasma should be the best test for detection of pheochromocytoma, since the kidney can directly alter the excretion of metabolites.
Because plasma concentration of free normetanephrine is low and the presence of inhibitors for the enzyme, normetanephrine in plasma cannot be detected. In a particular embodiment, conjugated normetanephrine is deconjugated (liberated) by acidic hydrolysis. Furthermore, a substantial degree of inhibition is overcome by decreasing the amount of incubated plasma to less than 100 microliters. The method is specific and rapid, yielding results in less than three hours while one person could easily perform thirty-six assays in eight hours. The method has particular importance with respect to the detection of normetanephrine in hypertensive patients even during therapy, as no antihypertensive drugs interfere with the assay.
Specifically, a method is provided for assaying an N-methylatable phenylethanolamine in a sample by converting the phenylethanolamine to its N-methylated derivative with a tritiated methyl group, and measuring the radioactivity of the derivative. Conversion can be accomplished by transmethylation, exemplified by reaction with S-adenosyl-L-methionine having a tritiated methyl group and promoted by a transfer enzyme therefor, exemplified by phenylethanolamine-N-methyl-transferase. The target phenylethanolamine typically would be found among a plurality of other N-methylatable phenylethanolamines. Accordingly, the method includes the step of separating the methylated target phenylethanolamine from other N-methylated phenylethanolamines prior to measuring the radioactivity. In a particular embodiment, the separation step comprises isolating the methylated target phenylethanolamine by thin layer chromatography. In a further embodiment, calculation is aided by concurrently processing a duplicate sample but to which has been added a known amount of the target phenylethanolamine, serving as a standard for the calculation. The isolated target derivative can then be combined with a scintillation fluid and its radioactivity measured in liquid scintillation counter.
In a particular embodiment, normetanephrine is assayed in a sample of plasma or urine containing a plurality of other N-methylatable phenylethanolamines. To the sample is added S-adenosyl-L-methionine having a tritiated methyl group, and phenylethanolamine-N-methyl transferase as promoter. The combination is incubated for a time sufficient to transmethylate the normetanephrine with the tritiated methyl group to form tritiated metanephrine. The specificity of the assay is based on the fact that the N-methylated phenylethanolamines have coefficients of extraction and chromatographic characteristics which differ one from the other. Accordingly, the tritiated metanephrine can be separated from other N-methylated phenylethanolamines and its radioactivity measured.
The technique presented by this disclosure is very convenient both to the patient and to laboratory personnel. For example, urine can be collected in any clean container without any preservative and an aliquot can be stored in a freezer until analysis. The assay allows collection of a random urine sample when the patient is hypertensive or symptomatic, with the analysis being conducted even several days later. The assay is sensitive enough to be used as a routine test. For example, one can measure less than 50 picograms of normetanephrine with a variability of about 3.5%, thus providing a specific, rapid and accurate assay for the detection of pheochromocytoma.
These and other objects and advantages of the invention, as well as the details of specific examples, will be more fully understood from the following: